Processes controlled by cGMP function to coordinate relaxation of multiple forms of smooth muscle including vascular tissue and airways. While cGMP levels in tissues are regulated by its biosynthesis through membrane bound and cytosolic or soluble forms of guanylate cyclase and by its removal by phosphodiesterases, stimulation of cGMP production by activation of the sGC is generally the most dominant mechanism involved in physiological processes that initiate smooth muscle relaxation through cGMP. Nitric oxide (NO) is one of the best understood activators of sGC (2, 12, 23, 33), but there are many other mechanisms that appear to control the activity of sGC. For example, the ROS superoxide anion is formed as a result of multiple disease processes, and it prevents NO from stimulating sGC by converting it to peroxynitrate (ONOO−). It also appears that superoxide, and oxidation of sGC thiols (25) and heme (11) sites prevent sGC activation by NO, and perhaps other stimuli. Asthma and most diseases altering the function of both vascular and non-vascular smooth muscle are known to be associated with increasing the activity of oxidases which are thought to produce reactive oxygen species (ROS) and reactive NO-derived species such as ONOO−. Thus, increased oxidative stress in asthma and vascular diseases appear to impair cGMP-mediated bronchodilator and vasodilator mechanisms through the attenuation of mechanisms involved in sGC stimulation by NO and other processes. Since smooth muscle growth and inflammation are promoted by oxidant mechanisms and these mechanisms are inhibited by cGMP, alterations in these mechanisms may also contribute to the inflammation and remodeling that is seen in asthma, and vascular diseases, and other smooth muscle systems such as bladder dysfunction. The cGMP system is also thought to promote angiogenesis which will contribute to the restoration of tissue perfusion needed to treat complications of diabetes.
It was found that sGC purified from bovine lungs was stimulated by low nanomolar concentrations of the biosynthetic iron-free precursor of heme, protoporphyrin IX, in the process of elucidating how NO activates sGC (19, 34). However, it was not demonstrated that protoporphyrin IX could function as a regulator of sGC and relaxation in smooth muscle presumably because of the poor tissue permeability of this porphyrin molecule. The ability of protoporphyrin IX to stimulate sGC would not be impaired by oxidation of heme because it does not contain iron. In addition, the thiols that regulate sGC activation appear to be at a site that enhance NO binding to heme, and not at the actual heme/protoporphyrin IX binding site on sGC. Thus, if sGC activity in smooth muscle were activated by protoporphyrin IX, it might be resistant to inhibition by oxidant stress, and its mechanism could be developed as an approach to promote smooth muscle relaxation and other beneficial actions of cGMP under the oxidative stress-associated conditions present in asthma, vascular diseases and other disorders altering smooth muscle function.
NO activates sGC by binding a ferrous heme group, which appears to be a cofactor that is normally bound to sGC when it is isolated from tissues (6, 9, 34). Early studies investigating how NO activated sGC associated with the initial discovery that the iron-free biosynthetic precursor to heme, protoporphyrin IX activated sGC provided evidence for how sGC was activated (19). The protoporphyrin IX-activated form of sGC showed changes in enzyme kinetic properties (e.g. KM for Mg-GTP and maximum velocity) which resembled the NO-stimulated form of sGC (34). This observation resulted in the proposal that NO stimulated sGC by binding the iron of the heme of sGC resulting in a disruption of the bond normally present between an amino acid of sGC and the iron of the sGC heme (34). This amino acid was subsequently identified as a histidine residue (5, 31). Heme oxygenase-derived carbon monoxide has been reported to activate guanylate cyclase (4, 14) and causes vascular relaxation associated with increases in cGMP (14). Cleavage of the histidine bond to heme by NO distinguishes the marked stimulation of sGC activity by NO from a modest activation by CO (31), which does not result in the cleavage of the iron-histidine binding interaction (5). None of the other biosynthetic precursors of heme other than protoporphyrin IX made within mitochondria were found to alter sGC activity (19), and heme was observed to be a competitive inhibitor of the activation of sGC by protoporphyrin IX, suggesting both porphyrins bound to the same site on sGC (34). While drugs which mimicked human diseases of porphyrin metabolism associated with increased hepatic levels of protoporphyrin DC were observed to increase cGMP levels in liver (20), minimal work has been done to investigate the biological significance of this mechanism of regulating sGC, or if increasing cellular protoporphyrin IX levels could be used as a therapeutic approach. This may be because one would not expect protoporphyrin DC to be released from mitochondria in amounts that activate sGC, an enzyme known to be located in the cytosolic region of cells. Applicants have now unexpectedly found that protoporphyrin IX can be released from mitochondria in the amount sufficient to activate sGC.
The accumulation of protoporphyrin IX generated from the heme precursor δ-aminolevulinic acid (ALA) has been investigated as an approach to detect tumor cells based on its fluorescence and as a phototherapy. The scientific literature in this field documents that ALA is easily delivered to human patients and animals in amounts that promote detectable protoporphyrin IX accumulation. While measurements of protoporphyrin IX fluorescence in tumor and normal cells also detects fluorescence from its biosynthetic precursors uroporphyrin III and coproporphyrin III, the ratios of these porphyrins appears to be similar across a variety of cell types (27). Thus, tissue protoporphyrin IX fluorescence after treatment with ALA has been demonstrated in many studies to be closely associated with measurements of its levels in tissue extracts (30). The photosensitizing action of the protoporphyrin IX accumulated during exposure to ALA has also been developed as a phototherapy approach to kill tumors and other proliferating cells. A reduction in systemic and pulmonary artery pressure was reported as a factor which limited the doses of ALA that could be used in humans in this approach (13). However, the mechanism by which ALA directly or indirectly mediates these effects has not been elucidated. In addition, phototherapy using ALA administration to generate protoporphyrin DC has been investigated as an approach to treat restenosis through its cell death promoting actions (24, 28). Studies examining the treatment of animals with ALA for restenosis phototherapy have documented that ALA increases protoporphyrin IX fluorescence levels in the smooth muscle region of the arterial wall (21, 28). Treatment of isolated skeletal muscle arterioles with ALA has been used in studies on vascular regulation by the heme oxygenase system as a method of increasing heme availability for the production of carbon monoxide (22). However, there do not appear to be studies examining the regulation of sGC resulting from protoporphyrin IX generation by ALA in vascular tissue or any other cellular system.